Protein folding cannot be understood without knowing the structural basis for folding energetics. The long-term goal of these studies is to provide such an understanding for ?-sheets-one of the three most common secondary structures in folded proteins (the other two being ?-helices and reverse turns). While substantial progress toward this goal has been made over the lifetime of this project, important questions remain, including the influence of co- and post-translational modifications on ?-sheet folding energetics. N-glycosylation is a co-translational modification occurring on about 1/3 of the secreted eukaryotic proteome before folding commences. In the last cycle we demonstrated that: (1) the stabilizing effects of N-glycosylation on ?-sheet folding stem largely from specific protein-glycan interactions within a reverse turn structural motif called an enhanced aromatic sequon, which can be engineered into a substantial fraction of proteins; and (2) the oligosaccharyltransferase enzyme complex (OST) prefers to glycosylate proteins with enhanced aromatic sequons. Now we propose to explore the contributions of the glycan to native state stabilization. Our preliminary results suggest that many natural and unnatural monosaccharides attached to the Asn side chain nitrogen can enhance ?-sheet folding energetics better than N-acetylglucosamine (GlcNAc), the sugar attached to Asn by OST in the cell. We hypothesize that super-stabilization of small proteins by unnatural N-glycosylation could render more proteins pharmacologically useful and more enzymes industrially suitable. In Specific Aim 1, we will determine the generality and scope of native state stabilization by N-glycosylating enhanced aromatic sequons with monosaccharides and disaccharides other than GlcNAc and GlcNAc-GlcNAc in the Pin WW domain and two pharmacologically interesting proteins. Since the second sugar often makes longer-range interactions within the protein-of-interest, we hypothesize that optimizing this interaction can be utilized to slow the unfolding rate and reduce aggregation, which leads to loss of function. In Specific Aim 2, we will stabilize antibodies via a engineered enhanced aromatic sequon using natural N-glycans. Preliminary results show that engineering an enhanced aromatic sequon into the CH2 domain in the context of a type IV ?-turn-a new enhanced aromatic sequon-stabilizes the Fc fragment of an IgG-type antibody. A crystal structure shows that the n-2 Phe residue makes stabilizing interactions with both the GlcNAc and a fucose residue ?1,6-linked to GlcNAc. We will also determine how this stabilizing modification influences the effector functions of the Fc fragment. The proposed work is significant because we will generate engineering rules to substantially stabilize native states with slowed unfolding, which should translate into improved protein drug serum half-lives and decreased aggregation during storage. The research proposed is innovative because we utilize a common post-translational modification, both as it occurs in nature and in chemically optimized forms, as a platform to engineer new, highly stabilizing interactions into ?-sheet-rich protein native states.